The yeast, Saccharomyces cerevisiae, offers a good system for the study of eukaryotic DNA replication. Its chromosomal DNA replication is similar to that in higher eukaryotes in that initiation sites are internal and chromosomes contain more than one replication unit. In contrast to the situation in higher eukaryotes, its chromosomal DNA is small enough to be isolated and examined by physical techniques without shearing and DNA replication mutants are available. In this proposal I outline experiments involving electron microscopic observation of intact, replicating DNA molecules which will provide a replication map of several yeast chromosomes and which answer the questions of (1) whether initiation sites are specific, and (2) whether the temporal sequence of replication is constant. Other experiments are outlined which will determine whether yeast DNA replication is unidirectional or bidirectional, whether any of the DNA replication mutants are blocked at stages of replication identifiable by electron microscopy. Density transfer and isotopic labeling experiments are described which will determine when during the cell cycle mitochondrial DNA is synthesized, whether nuclear and mitochondrial DNA share replication enzymes and whether the amount of mitochondrial DNA per cell is regulated.